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Team:NTNU Trondheim/Protocols
From 2014.igem.org
(Difference between revisions)
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<div style="top:55px">Media</div> | <div style="top:55px">Media</div> | ||
</li> | </li> | ||
| - | <li id=" | + | <li id="week1" onclick="weekFilter(this)"> |
<a class="nb-week" href="#1">Liquid Broth (LB)</a> | <a class="nb-week" href="#1">Liquid Broth (LB)</a> | ||
</li> | </li> | ||
| - | <li id=" | + | <li id="week2" onclick="weekFilter(this)"> |
<a class="nb-week" href="#2">Super Optimal Broth (S.O.B.)</a> | <a class="nb-week" href="#2">Super Optimal Broth (S.O.B.)</a> | ||
</li> | </li> | ||
| - | <li id=" | + | <li id="week3" onclick="weekFilter(this)"> |
<a class="nb-week" href="#3">yB medium</a> | <a class="nb-week" href="#3">yB medium</a> | ||
</li> | </li> | ||
| - | <li id=" | + | <li id="week4" onclick="weekFilter(this)"> |
<a class="nb-week" href="#4"><i>Synechocystis</i> medium</a> | <a class="nb-week" href="#4"><i>Synechocystis</i> medium</a> | ||
</li> | </li> | ||
| - | <li id=" | + | <li id="week5" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#5"></a> |
</li> | </li> | ||
<li class="nb-month"> | <li class="nb-month"> | ||
<div style="top:140px;margin-left:-18px">Techniques</div> | <div style="top:140px;margin-left:-18px">Techniques</div> | ||
</li> | </li> | ||
| - | <li id=" | + | <li id="week6" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#6">Gibson assembly</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week7" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#7">DNA isolation and cleaning</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week8" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#8">DNA digestion</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week9" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#9">PCR</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week10" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#10">NanoDrop</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week11" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#11">3A assembly</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week12" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#12">Ligation</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week13" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#13">Transformation (<i>Escherichia coli</i>)</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week14" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#14">Transformation (<i>Synechocystis sp. PCC 6803</i>)</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week15" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#15">Gel electrophoresis</a> |
</li> | </li> | ||
<li class="nb-month"> | <li class="nb-month"> | ||
<div style="top:52px;margin-left:-9px">Plasmids</div> | <div style="top:52px;margin-left:-9px">Plasmids</div> | ||
</li> | </li> | ||
| - | <li id=" | + | <li id="week16" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#16">Right flank</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week17" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#17">Left flank</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week18" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#18">Kanamycin</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week19" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#19">Lac inducible promoter</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week20" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#20">Glucose oxidase</a> |
</li> | </li> | ||
<li class="nb-month"> | <li class="nb-month"> | ||
<div style="top:52px;margin-left:-22px">Organisms</div> | <div style="top:52px;margin-left:-22px">Organisms</div> | ||
</li> | </li> | ||
| - | <li id=" | + | <li id="week21" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#21"><i>Escherichia coli</i> DH5α</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week22" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#22"><i>Synechocystis sp. PCC 6803</i></a> |
</li> | </li> | ||
<li class="nb-month"> | <li class="nb-month"> | ||
<div style="top:55px;margin-left:-29px">Calculations</div> | <div style="top:55px;margin-left:-29px">Calculations</div> | ||
</li> | </li> | ||
| - | <li id=" | + | <li id="week23" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#23">Transformation efficiency</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week24" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#24">Enzyme amount</a> |
</li> | </li> | ||
| - | <li id=" | + | <li id="week25" onclick="weekFilter(this)"> |
| - | <a class="nb-week" href="# | + | <a class="nb-week" href="#25">DNA concentration</a> |
</li> | </li> | ||
</ul> | </ul> | ||
Revision as of 12:43, 8 August 2014
Team:NTNU Trondheim/Protocols
From 2014.igem.org
"
Team:NTNU_Trondheim/Protocols
From 2014.igem.org
Jump to:
-
Media
- Liquid Broth (LB)
- Super Optimal Broth (S.O.B.)
- yB medium
- Synechocystis medium
-
Techniques
- Gibson assembly
- DNA isolation and cleaning
- DNA digestion
- PCR
- NanoDrop
- 3A assembly
- Ligation
- Transformation (Escherichia coli)
- Transformation (Synechocystis sp. PCC 6803)
- Gel electrophoresis
-
Plasmids
- Right flank
- Left flank
- Kanamycin
- Lac inducible promoter
- Glucose oxidase
-
Organisms
- Escherichia coli DH5α
- Synechocystis sp. PCC 6803
-
Calculations
- Transformation efficiency
- Enzyme amount
- DNA concentration
Protocols
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Media
Techniques
Plasmids
Organisms
Calculations
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Liquid Broth (LB)
Recipe
Antibiotic additions
| Antibiotic | Stock concentration | Final concentration | Dillution factor | Solvent | Storage temperature |
|---|---|---|---|---|---|
| Ampicillin | 50 mg / mL | 50 μg / mL | 1000 | Filter sterilized H2O | 4 °C |
| Chloramphenicol | 30 mg / mL | 30 μg / mL | 1000 | Ethanol | -20 °C |
| Kanamycin | 50 mg / mL | 30 μg / mL | 1000 | Filter sterilized H2O | 4 °C |
| Spectinomycin | 50 mg / mL | 50 μg / mL | 1000 | Filter sterilized H2O | 4 °C |
Ingredients:
- Tryptone (10g)
- NaCl (10g)
- Yeast Extract (5g)
- Fill with 1 L of distilled / filtered H2O.
- Autoclave at 121 °C for 20 minutes.
- Add antibiotics if needed, after the medium has cooled down.
Super Optimal Broth (S.O.B.)
Recipe
show technical details
{{{tech}}}
Made LB plates with ampicillin and ampicillin + kanamycin.
Gibson Assembly
Protocol from New England Biolabs
show technical details
{{{tech}}}
- Set up the following reaction on ice:
- Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section). - Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μL of the assembly reaction, following the transformation protocol.
| Recommended Amount of Fragments Used for Assembly | |||
| 2-3 Fragment Assembly | 4-6 Fragment Assembly | Positive Control** | |
| Total Amount of Fragments | 0.02–0.5 pmols* X μl |
0.2–1 pmols* X μl |
10 μl |
| Gibson Assembly Master Mix (2X) | 10 μl | 10 μl | 10 μl |
| Deionized H2O | 10-X μl | 10-X μl | 0 |
| Total Volume | 20 μL*** | 20 μL*** | 20 μL |
** Control reagents are provided for 5 experiments.
*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
Gibson Assembly
Protocol from New England Biolabs
show technical details
{{{tech}}}
- Set up the following reaction on ice:
- Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section). - Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μL of the assembly reaction, following the transformation protocol.
| Recommended Amount of Fragments Used for Assembly | |||
| 2-3 Fragment Assembly | 4-6 Fragment Assembly | Positive Control** | |
| Total Amount of Fragments | 0.02–0.5 pmols* X μl |
0.2–1 pmols* X μl |
10 μl |
| Gibson Assembly Master Mix (2X) | 10 μl | 10 μl | 10 μl |
| Deionized H2O | 10-X μl | 10-X μl | 0 |
| Total Volume | 20 μL*** | 20 μL*** | 20 μL |
** Control reagents are provided for 5 experiments.
*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
